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Forensics in HIV Transmission Crimes
Investigación Forense en Crímenes de Transmisión del VIH
Grazinoli Garrido Rodrigo
1
; Gonçailves da Costa Gabriela
2
1- Biomédico; Grad. Segurança Pública; MSc; DSc Perito Criminal - Diretor do IPPGF/PCERJ Professor Adjunto -
PPGD/UCP - FND/UFRJ. ORCID ID https://orcid.org/0000-0002-6666-4008 2- Biomédica
MSc. Rodrigo Grazinoli Garrido - grazinoli.garrido@gmail.com
Recibido: 21-09-2019 Aceptado:15-I-2020
Resumen
La microbiología forense es un área científica que ha surgido con la necesidad de investigar los delitos
biológicos, como en el caso de la transmisión intencional del virus de la inmunodeficiencia humana (VIH).
Este trabajo exploratorio tuvo como objetivo demostrar cómo la tecnología biomédica, como la filogenética
y la cuantificación de la carga viral y los linfocitos T CD4+, puede usarse para producir evidencia técnica
que brinde más certeza para determinar la autoría y la materialidad de estas conductas criminales.
Palabras claves
Investigación forense; SIDA, VIH, crimen.
Fuente: DeCS
Abstract
Forensic microbiology is a scientific area that has emerged with the need to investigate biocrimes, as in the
case of intentional transmission of the Human Immunodeficiency Virus (HIV). The present exploratory
work aimed to demonstrate how biomedical technology, such as phylogenetics and quantification of viral
load and CD4+ T lymphocytes, can be used to produce technical evidence that brings more certainty in
determining the authorship and materiality of these criminal behaviors.
Key words
Forensics; AIDS; HIV, crime
Source: DeCS
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Introduction
With advances in microbiology in recent years, forensic microbiology has been established and strengthened
as a new scientific chair for responding to the law at a biological crime event, this is a discipline in which
microbiology and forensic science complement each other and are dedicated to tracking and analyzing a
biocrime.
The need for the study of microbiological expertise can be applied in biocrimes linked to the transmission
of microorganisms intentionally, such as the intentional transmission of Human Immunodeficiency Virus
(HIV), a pathogen that causes Acquired Immunodeficiency Syndrome (AIDS).
Many issues arose with this pandemic, one of them was the need on the part of jurists to solve the difficult
and complex framework, which affects not only the criminal sphere but also the social and cultural sphere,
the conduct of the individual who commits this crime.
The jurisprudence and the doctrine still maintain some disagreement about the possible typifications of
crimes involving intentionality in the transmission of the virus. The position of the United Nations Joint
Program on HIV / AIDS, to avoid further discrimination against carriers, is to avoid establishing legislation
to address the issue or a specific criminal type. Thus, countries such as Brazil seek to frame criminal conduct
in broader criminal types.
However, whatever criminal type the conduct may fall under, it is certain that for the perfect characterization
of the crime, it is necessary to produce technical evidence based on current biomedical knowledge and
technologies. Thus, it would be possible to establish the causal and temporal connection between the
conduct and the outcome.
The current research was developed through exploratory and qualitative analysis, based on indirect
documentation from national secondary sources. We sought to analyze new technologies applicable to the
determination of causality and temporality between certain suspicious behaviors and effective
contamination and prognosis of the victim's disease, highlighting the use of viral phylogenetics.
HIV: General Aspects
HIV is grouped into the genus Lentivirus (lentus, from Latin) due to the slow course of infection and thus
disease, with a long latency period, persistent viral replication and central nervous system involvement (1).
Regarding the family, it is grouped within the Retroviridae family, viruses that have the enzyme Reverse
Transcriptase (TR) - responsible for transcribing the RNA genome into complementary DNA (cDNA),
being the subfamily Orthoretrovirinae (2). This family of retroviruses has been receiving a lot of attention
from scientists in recent decades for causing serious diseases in humans, such as AIDS (3).
HIV is divided into two types, HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV-1 has worldwide
distribution, while HIV-2 is more frequently detected in individuals from African countries (4).
The first clue to the emergence of HIV-2 came in 1986 when a morphologically similar but previously
distinct virus was found to cause AIDS in patients in West Africa (4). This new virus, described as HIV-2,
was closely related to a virus that caused immunodeficiency in captive monkeys in sub-Saharan Africa. The
microorganisms, isolated in these animals, were collectively called Simian Immunodeficiency Virus (SIV),
are also grouped with the genus of Lentivirus (4).
Interestingly and surprisingly, these viruses appeared to be largely non-pathogenic in their natural hosts, the
primates, since ape viruses familiar with HIV-1 and HIV-2 were found infecting these animals. These
relationships provided the first evidence that AIDS emerged in humans and monkeys as a consequence of
infections between different primate species (4). HIV-1 evolved from nonhuman primates, the Central
African chimpanzees, which were infected with the Central African Human Immunodeficiency Virus
(SIVcpz) and West African mangrove HIV-2 (SIVsm) (5). Thus, it was clear that HIV-1 and HIV-2 were
the result of zoonoses from primate-infected virus transfers in Africa (6).
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This viral barrier breakdown occurred through hunting activities, which caused humans to acquire SIV (7).
However, SIV is a weak virus, typically suppressed by the human immune system within weeks of infection.
It is believed that several transmissions, from the simian immunodeficiency virus, from individual to
individual in rapid succession have enabled its transformation into HIV over time (8).
HIV infection is said to be chronic or persistent since the infected host is unable to eliminate the infectious
agent (9). Most infections by this virus occur through the mucous membranes of the genital or rectal tract
during sexual intercourse, but there are other ways of transmission of this pathogen, besides the sexual route,
such as parenteral and vertical (10).
After exposure to HIV, if infection occurs, there are approximately ten days called the eclipse phase, before
viral RNA is detectable in plasma (11). After this moment, the pathogenic pathway of this infection goes
through three main and sequential stages: initial or primary phase, asymptomatic or clinical latency phase,
and symptomatic phase (10), as shown in Figure 1.
Figure 1 - Natural history of HIV infection in the absence of antiretroviral therapy (58).
Primary infection, or acute viral syndrome, is defined as the period between the initial infection and the
development of the immune response and lasts no longer than two or three weeks (10). In general, at this
stage, HIV-infected individuals may develop asymptomatically, presenting with a clinical picture similar to
influenza or mononucleosis-like (exhibiting signs and symptoms typical of these conditions - fever, myalgia,
malaise, headache, nausea, vomiting, pharyngitis, among others) (12).
The innate immune response established at the focus of the infection attracts an additional amount of T
cells, which causes intense viral replication (with viremia values up to 108 copies of RNA / mL of plasma).
With the visceral and lymphoid tissue dissemination, which are targets of the action of this pathogen, and
because of this, there is a marked decrease in CD4+ T lymphocytes and an absence of immune response on
the part of the host, thus ensuring a significant viremia and Antigenemia. These will gradually decrease as
the patient develops an immune response to HIV (9, 11).
Importantly, infection with this virus has an immunological window time of approximately 30 days. This
phase is characterized by the period between infection and the body's production of antibodies against the
viral particle in sufficient quantities to be detected by the tests (13). In this interval, the person may already
be infected and still have the antibody test result as negative (14).
After primary infection follows the clinical or asymptomatic latency phase. At this time clinical recovery
occurs, with a reduction of viral replication as a result of the immune response, but it is insufficient in
magnitude to eradicate the infection. It is at this stage that seroconversion occurs, with the development of
antibodies that persist throughout the body (9). Several factors may be implicated in the control of viral
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replication, including the presence of neutralizing antibodies and cytotoxic T cells. These exert partial
control of infection, but not sufficiently to prevent, in the absence of therapy, the slow and progressive
depletion of CD4+ T lymphocytes. and the eventual progression to AIDS (11). In general, it can be said that
at this stage a balance is struck between viral replication and host immune response (5).
The asymptomatic phase is therefore characterized by reduced viral loads due to the strong immune response
of the host, as there is an additional production of activated CD4+ T lymphocytes that target new infections,
and the absence of clinical symptoms and signs of the disease (11). The time between initial infection and
the development of clinical disease varies considerably, with an average of ten years (9).
Finally, in the symptomatic phase, there is intense viral replication and decreased immune response due to
the gradual decrease of TCD4 lymphocytes throughout the asymptomatic phase. This favors the appearance
of neoplasms and opportunistic infections of increasing severity and lethal potential. This period may last a
few months or several years (9).
In the advanced stage of HIV infection, infected individuals may progress to AIDS. However, for this to be
defined, the patient must have opportunistic infections (such as pneumocystosis, neurotoxoplasmosis,
atypical or disseminated pulmonary tuberculosis, cryptococcal meningitis, and cytomegalovirus retinitis)
and neoplasms (the most common being Kaposi's sarcoma, non-Hodgkin's lymphoma and cervical cancer
in young women). In these situations, the CD4+ T lymphocyte count is below 200 cells/mm³, most of the
time and there are high levels of circulating viruses (15).
As a therapeutic strategy about HIV, there are antiretrovirals (ARVs). These drugs appeared in the mid-
1990s to prevent the virus from multiplying in the body. Before the emergence of this type of therapy,
clinical management of HIV consisted largely of prophylaxis against common opportunistic pathogens and
the management of AIDS-related diseases (16).
ARVs do not destroy the viral particle but prevent its replication, thus helping to prevent the weakening of
the immune system. Therefore, its use is fundamental to increase the time and quality of life of those living
with HIV / AIDS (14).
There are some classes of ARV drugs, which act at different sites on the viral particle. In general, drugs of
two classes are combined to ensure a potential attack on HIV. This strategy aims to prevent resistance to
ARVs by the pathogen. The advent of combination therapy, also known as HAART (Highly Active
Antiretroviral Therapy) for the treatment of HIV infection, particularly HIV-1, has been seminal in reducing
associated morbidity and mortality. HIV infection and AIDS. HAART dramatically suppresses viral
replication and reduces HIV plasma viral load below detection limits of the most sensitive clinical trials
(<50 RNA copies/ml), resulting in a significant immune system reconstitution as measured by an increase
in CD4+ T lymphocytes circulating (16).
The seven major classes of ARVs currently used for HIV treatment are: Fusion Inhibitors (IF), Integrase
Inhibitors (II), Protease Inhibitors (PI), Nucleoside Analogs Reverse Transcriptase Inhibitors (NRTI),
Reverse Transcriptase Inhibitor (NtRTI) Nucleotides, Non-Reverse Transcriptase Inhibitor (NNRTI)
Analogs, and Maturation Inhibitors (MI) (17).
The classes of ARVs that target reverse transcriptase act by inhibiting the action of this enzyme that acts on
the synthesis of HIV genetic material. Protease inhibitors impede the processing of viral proteins, leading
to the formation of defective viral particles unable to assemble the other complete virus. Besides, there are
also virus-cell inhibitors, which fall into two groups: fusion inhibitors and CCR5 inhibitors. For the use of
CCR5 inhibitors, it is important to verify if the infection is by viruses that present this surface protein.
Integrase inhibitors, on the other hand, prevent viral genetic material from integrating with cell DNA and,
finally, maturation inhibitors make the newly formed virus lack infectious capacity (18).
Currently, a relevant issue associated with HIV, which is a means of preventing infection by this virus is
Pre-Exposure Prophylaxis (PrEP), which is highly effective and was initiated in Brazil at the end of 2017
(19). Brazil is at the forefront of using PrEP as a means of prevention in Latin America, being the only
country in this region where PrEP is available through the public sector. PrEP, also known as “combined
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prevention”, is a combination of two antiretrovirals (tenofovir/emtricitabine) taken every day by people at
high risk for HIV, such as serodiscordant couples and people using injecting drugs. Daily use of this
medicine reduces the risk of contracting HIV by sex by over 90% and in people who use injecting drugs by
over 70% (20).
However, it is necessary for the individual who uses this method to perform HIV testing every three months
(19). It is noteworthy that PrEP does not prevent other sexually transmitted infections (STIs) or pregnancy,
so the importance of maintaining the use of other physical means of prevention, such as condoms. Another
feature of this prophylactic medium is that its efficacy is greatly reduced by the lack of compliance with the
daily dosage (10). PrEP is expensive (although generally free after application to the pharmaceutical
program), requires engagement in a health center and frequent follow-up visits (10).
Post-Exposure Prophylaxis (PEP) means taking antiretroviral drugs after being potentially exposed to HIV
to prevent infection. OPEP (Occupational Post-Exposure Prophylaxis) was used as a model for the creation
of Non-Occupational Post-Exposure Prophylaxis (nPEP), and in many respects they are similar, but for
oPEP, in most cases, there is more chances of testing the source of infection (20).
In both oPEP and nPEP, antiretroviral therapy is administered when an individual has been exposed to a
suspected or positive HIV secretion of semen, vaginal fluid or blood and seeks medical treatment within 72
hours and continued for 4 weeks. This prophylactic regimen has low side effects and minimal risk of HIV
resistance. However, if the patient's source is HIV negative or 72 hours have passed, PEP is not
recommended (19).
One fact that should be considered regarding HIV infection is that although cure for this pathogen is not yet
a current reality, an important issue is that after a decade of research, there is scientific evidence-based
confirmation that the risk of HIV transmission from a person living with HIV / AIDS (PLWHA) who is on
antiretroviral therapy (ART) and has achieved an undetectable viral load on blood for at least six months is
nonexistent. Being undetectable does not mean that the virus is no longer circulating in the blood, but it is
so low that it is not detected by the viral load test (14).
In HIV-related criminal cases, two points should be considered: the amount of virus in the suspect's
bloodstream; the immune response of the individual's organism; the nature and efficacy of the therapy
performed, considering that it is aware of its seropositive status. Therefore, viral load and CD4+ T
lymphocyte count are crucial in this investigation (21).
The CD4+ T lymphocyte count measures the number of this cell line by the flow cytometry technique. Since
CD4+ T lymphocytes are the target of HIV, these cells are progressively destroyed and the lower the count,
the more the disease progresses and the worse the symptoms. Counting these T cells does not check for
HIV, but rather measures the immune system response. Conversely, when the CD4+ T lymphocyte count is
high, viral replication is lower. If this occurs over a prolonged time, a long-term nonprogressive individual
is characterized. Treatment, risks, and prognosis of seropositive patients will be influenced by CD4+ T cell
count (22).
To properly determine viral load, amount of virus in the bloodstream, viral RNA is counted. The
quantification of the genetic material of the virus is performed by molecular methods, being a more accurate
and direct way of measuring the virus. As already stated, this measure correlates with the response to therapy
and can predict the progression of AIDS and how much HIV poses risk to the patient and their sexual
partners (22).
It is noteworthy that some HIV-positive individuals may have undetectable levels of the virus; in general,
patients with these characteristics are those who use antiretroviral therapy effectively (22). However, even
patients considered undetectable in a case of intentional transmission of HIV cannot be excluded from
prosecution, even with considerably lower chances of contamination (21). All existing knowledge about
HIV should be considered in expertise on a possible crime of HIV transmission.
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3. TECHNICAL EVIDENCE PRODUCTION: PHYLOGENETIC ANALYSIS
Forensic science is the key to establishing links between evidence found at a crime scene and suspects linked
to it (23), the application of modern techniques to identify or specify evidence in a judicial process is
essential (24).
Phylogenetic study has often been used as a forensic technique to investigate whether the relationship
between the types of HIV viruses that infect a set of individuals is compatible with the form of transmission
between them, thus whether it has occurred directly or indirectly (25).
Molecular phylogenetics, a field of phylogenetics, is a disciplinary study of the evolutionary relationships
between organisms using molecular sequences. The methods of analysis used in molecular phylogenetics
were originally developed to reveal evolutionary pathways. Even today this area is used in various fields,
such as biology and biodiversity (26), molecular epidemiology (27, 28), identification of gene functions
(29) and identification of microorganisms in microbiome studies (30, 31).
A phylogenetic tree or phylogeny is a graphical representation of ancestral-descendant relationships
between organisms or genetic sequences and should be considered as a hypothesis of an evolutionary
relationship between a group of organisms (32). They show evolutionary relationships between a set of
Operational Taxonomic Units (OTUs) (33).
The OTU usually represents a species, but can also represent individual organisms in a population, a protein
gene or sequence, or a taxon in any taxonomic position (by family, order, class, phylum). The nodes at the
tips of the tree are called "outer nodes". They are used to represent OTUs. Another type of node, called
"internal nodes," represents a Recent Common Ancestor (RCA). These include the lines, called "branches,"
used to connect newer and older nodes and show the evolutionary relationships between taxa. A branch that
connects two inner nodes is an "inner branch" that shows an old relationship. On the other hand, the branch
that joins an inner node to an outer node to show a newer relationship is called an "outer branch." The
deepest branch of the tree represents the "root" (33).
Linked to the phylogenetic study is the use of sequencing as an instrument to achieve the final result, the
construction of the phylogenetic tree. The classical sequencing technique is based on making many copies
of a target DNA, being introduced into the terminator nucleotide polymerization reaction and thus producing
fragments of different lengths. Also, the "chain terminator" nucleotides are fluorescently labeled, allowing
the ends of the fragments to be determined. Thus, it is possible to organize the various fragments by size
and perform sequencing by the terminal bases of each (34).
This technique was described by British biochemist Frederick Sanger and colleagues (35) and is thus called
Sanger Sequencing. Currently, however, it is possible to perform individual human sequencing in less than
one day, more cheaply (34,35,36). Therefore, Next Generation Sequencing (NGS), in which small
sequencing reactions can be conducted in parallel, has been used. Thus, large amounts of DNA can be
sequenced, increasing the speed and reducing the costs of the technique and the time to perform it. NGS is
a technique that favors high accuracy in results, which is of paramount importance, especially within the
forensic application (37).
The defining characteristic of HIV is its exceptional genetic diversity. This high diversity derives from at
least four sources: high substitution rates, a very small genome, short generation times and high
recombination frequency (38).
Through the progress of this method, it was possible to expand the study of HIV genetic diversity,
evolutionary and epidemic processes, improving the characterization of this virus and the possibility of
generation of complete virion genomes. In addition to greater sensitivity and accuracy in detecting
recombinant viral forms and detecting multiple viral infections in individual hosts (39). Another relevant
aspect related to this method is the possibility of detecting HIV variants at a low frequency, about 1%, and
it is potentially possible through this detection to specify information about viral particle infection dates,
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predicting whether this is a recent infection or not, which in the criminal context may be important to
estimate the moment of infection (40,41).
Unlike human DNA, which remains stable for life, RNA, the genetic material of HIV, has a rapid rate of
evolution, because TR cannot edit the newly synthesized genome, an activity known as proofreading. Thus,
erroneous nucleotide pairings during DNA synthesis are not verified and errors are consequently
incorporated into the nascent DNA molecule, thus implying a high rate of incorporation of mutations by
this virus. Thus, it gives rise to a high genetic diversity, making this virus a “moving target”, which
contributes to the host's inability to control and eliminate this pathogen in natural infection (42).
Phylogenetic methods, therefore, are a source for determining patterns of routes that HIV travels in cases of
transmission between individuals (46). During transmission, one or a few of these virus variants are passed
on and subsequently diverge in newly infected individuals so that epidemiologically linked persons never
have the same viral type (47). Due to the enormous genetic variation of the virus, this trait has been
successfully used for epidemiological-scale research to understand the evolutionary history of HIV and
phylogeography (48), and for local scale to study transmission networks of HIV-positive patients, especially
those of HIV-1, because it is the most present in the population (49).
These studies are already used as evidence in court, confirming or refuting the transmission between the
defendant and the victim (40). Besides, the phylogenetic study can also be used to develop better public
health prevention initiatives for this pathology (49) or to study the transmission of drug resistance (50).
In criminal cases addressing allegations of HIV-1 transmission, the forensic scientific approach may be
required to prove the timing and direction of transmission to demonstrate that the defendant infected the
victim and that the accused was aware of the diagnosis. HIV positive at the time of the alleged transmission
and to confirm that the victim was infected at the time of the events described in the prosecution (40).
However, this evidence for individuals infected with HIV-1 is challenging, as each patient has a quasi-
species of rapidly evolving viral strains (25). Performing virus differentiation in two epidemiologically
linked individuals depends on many factors and cannot yet be reliably predicted (25).
For this reason, there are concerns regarding the use of phylogenetic analysis, particularly whether it may
indicate the meaning and timing of HIV transmission and whether intermediate links may be excluded (25,
51). Therefore, the use of this method in courts involves strict care, because it is difficult to know for sure
that all the people involved in the transmission network were sampled. Missing calls need to be evaluated
through contact tracking, and these depend on testimony from the defendant and victim, and potentially
other witnesses. Therefore, in reconstructing a history of transmitting a phylogenetic structure in the context
of forensic investigations, one should never assume that all links are known. Victims must remember or be
willing to fully disclose all risk contacts (40).
The results of the phylogenetic analysis should be interpreted with caution in a court and need to be placed
in the context of other types of evidence (40). A probative combination: forensic evidence related to DNA
molecules, evidence of intercourse between parties, and health history of both the accused and the victim
should also be analyzed. Phylogenetic inquiry is just one of the many steps required to frame what will be
concluded from the phylogenetic tree: that is, to understand whether the tree is consistent with or contradicts
the accusations (40).
It is noteworthy that treatment can prevent transmission (52), which is also relevant to determine if the
defendant was infectious at the time of the event (40).
ü Therefore, some scenarios (Figure 2) should be taken into consideration:
ü The victim was infected by the defendant, not the other way around;
ü There is a third individual with a similar viral strain, linking the defendant and the victim;
ü Both the victim and the defendant were infected with one or more similar third viral strains;
ü The victim was already HIV positive and was again infected with another strain, by the defendant or a
third party.
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Figure 2 - Hypothetical phylogenetic tree for investigation of HIV transmission (40).
In a study by Siljic and colleagues in the city of Belgrade, capital of Serbia, in 2011, three individuals were
analyzed as a possible source of intentional HIV transmission. The analysis reported one man (individual
1) and two women (individual 2 and individual 3), all diagnosed with HIV serology at the HIV / AIDS
Center of the University Hospital for Infectious and Tropical Diseases in August and September of that
year, in Belgrade (53).
The initial information regarding these contaminations was as follows:
ü Individual 1 and individual 2 had been married for more than 15 years with two children aged 10 and
14, both considered HIV negative;
ü Individual 3 had been a sexual partner with individual 1. Together, they had several extended stays in
Thailand in the years before HIV infection;
ü Individual 1 sues individual 3, alleging conscious contamination by the latter subject. And that
individual 3, according to individual 1, knew of his HIV-positive diagnosis and had not revealed it;
Sample analyses were performed using two genes, env, and pol. For the study, thirty-four sequences from
these same gene regions of different patients who had HIV-positive serology within two years before and
two years after the time of diagnosis of the three questioned patients were included in the phylogenetic
analysis, as local controls. Twenty-nine samples of the pol gene sequences of heterosexual, routine hospital
subtype B subjects who were resistant to drugs against the viral particle were also added. Because of possible
epidemiological linkages with Thailand, 20 subtype B viral sequences in pol and env sampled from those
infected in this region were also used, retrieved in the BLASTsearch (Basic Local Alignment Search Tool)
tool. The final dataset analyzed consisted of 101 local and 50 foreign sequences as controls for the pol gene
and 34 local and 50 foreign sequences as controls for the env gene (53).
As a result, the researchers noted that it was subtype B that infected the three subjects involved in the study.
Phylogenetic analyses were very consistent, showing that there was no mix of external control sequences in
the transmission cluster. And, the cluster contained no viral sequences from Thailand. As shown in Figures
3 and 4, there was a specific cluster for the three individuals analyzed without mixing of other viral
sequences (53).
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The most recent common ancestor time (tMRCA) provides an estimate for each subject's date of infection,
based on a 95% confidence interval of the root branch cluster's tMRCA estimate. For individual 1 the
estimated time was between 2003 and 2007 (for the pol gene) and 2006-2008 (under analysis of the env
gene), ie 3-8 years before the sampling date (2011). The estimated time for individual 2 was placed between
2007 and 2009 (in) and 2008-2010 (env), 1 to 4 years before the sampling / diagnosis date and for individual
3 between 2007 and 2009 (in) and 2008 -2010 (env), also 1 to 4 years before the diagnostic sampling date,
both presented the infection later than the individual 1(53).
This study reinforced the need to use adequate controls so that reliable results could be obtained at the end
of the analysis. To reach them, the researchers emphasized the importance of using viral sequences that
shared the same risk of transmission, similar geographic location, use of the same gene clades (subtype or
recombinant form) and that have proximity in the period of diagnosis of contamination. If sufficient samples
are included in the analysis as a control, this may indicate that the persons involved belong to a transmission
chain (53).
In conclusion, it was observed that there was a relationship between individuals 2 and 3, and individual 1 is
suggestive of being the source of infection for both, due to the estimation of tMRCA. This would refute the
a priori hypothesis of HIV transmission from individual 3 to 1, however, there is an epidemiological
relationship between the two. However, caution is still needed regarding the interpretation of potential court
findings (53).
Given this study, it is observed that the reliability of phylogenetic analysis to "prove" the transmission of
HIV between individuals should be approached as detailed as possible (25). Therefore, there is a need for
caution in producing technical evidence that should always be analyzed in it is a probative set, as it does not
have a superior hierarchical position in the judicial evaluation, but the technical tool enjoys great influence
in today's society since it carries the credibility of what is scientific (54).
Phylogenetic analysis is generally performed in research laboratory settings rather than in forensic
laboratories. Therefore, it is crucial to maintain the chain of custody, ensuring quality and care when
handling the analyzed samples to minimize the possibility of errors such as contamination and labeling and
in carrying out the scientific methods involved, which will be reported as an expert report and will be final
client to Justice (25, 55).
Figure 3 - Phylogenetic tree based on pol gene
sequences obtained in the study. The colored
sequences identify the individuals analyzed (53).
Figure 4 - Phylogenetic tree based on env gene
sequences obtained in the study. The colored sequences
identify the individuals analyzed (53).
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Another important issue is that if the chosen laboratory has no forensic experience, it is the applicant's task
to emphasize the importance of the double-blind test, ie the analyst performing the examination should not
be aware of the proposed transmission direction and the other circumstances of the case. Therefore, each
person's samples should be tested in two independent laboratories under “blind” conditions, thus eliminating
the possibility of laboratory error and investigator bias, thus yielding consistent results (25).
Sample tracking is also essential, so it is emphasized once again that chain of custody maintenance should
be given the highest priority, ensuring the authenticity and suitability of expert evidence, thus ensuring
evidence tracking from the crime scene to court, strict protocols on evidence being applied (56).
There are many ways to build a phylogenetic tree. Consideration should be given to the reliability of the
methods used for this training - including the HIV-specific genes analyzed - as well as the purpose of the
tree. It must be as impartial as possible. This includes choosing sufficient and adequate epidemiological
controls, analyzing approximately thirty other strains of HIV from individuals who are of the same
geographical origin, social context, and potential transmission network as defendant and victim(s) (25).
It has recently become common practice to also include publicly similar sequences selected as database
controls using BLAST search. At least ten control sequences must be added to search (40). These should
then be compared with the strains being investigated. Using inadequate controls may erroneously emphasize
any detected relationship between two viruses as being remarkably unique (25,53).
Also, controls must be collected at the time of the alleged broadcast event. This is crucial in building very
complex networks. In most cases, it will be difficult and often impossible to obtain samples from the
appropriate controls. As a result, the interpretation of the findings will need to be particularly cautious (25).
It is important to remember that similar strains can be found in more than two individuals if both are part of
a broader transmission network, which is very common among individuals with HIV. Consequently, even
with controls, phylogenetic analysis cannot "prove" transmission. However, when there is statistical support
to link the investigating individual closer to one of the controls rather than the complainant, the technique
is reliable enough to exclude the possibility of transmission (25).
It is understandable that in such a case the victim and society want a crime involving HIV infection to be
punished, however, no unfair conviction must occur in a court case. Therefore, forensic investigations with
phylogenetic and other evidence related to the transmission of this virus are potentially powerful to acquit
suspects (25, 57).
The issue of drug resistance due to virus mutations is generally removed from alignments before
investigations of HIV transmission in phylogenetic analyzes (25), as viral populations can respond quickly
and efficiently to environmental disturbances in which they replicate, offering a wide spectrum of mutations
on which natural selection can act (44). Therefore, when there are changes in the environment, for example
by administering antiviral drugs, the presence of one or more mutants more able to replicate in this new
medium causes the population derived from these mutants to gain resistance and increase their chances of
survival. In the case of HIV-1, selective pressure exerted constantly by the immune system results in the
virus adapting to new target cells and maintaining a persistent infection (44).
Everyone involved in the criminal justice system must be aware of the limitations of phylogenetic analysis
before using such evidence as conclusive or even suggesting HIV transmission between individuals, but all
results obtained must be of the highest quality (25, 57). Phylogenetic analysis can and does include some
degree of approximation and error (25). However, phylogenetic investigations have proven useful for
analyzing HIV transmission in forensic expertise, and many promising advances in research may enable its
use in future cases (40).
Conclusions
Forensic microbiology, despite being a new area within forensic science, is of great relevance for the
establishment of technical proof of crimes. In investigating the intentional transmission of HIV / AIDS it is
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111
possible to rely on phylogenetic methods, which traces the evolutionary relationship between the viral
strains of the victim and the suspect, revealing the path of infection and establishing the causal link between
conduct and outcome.
In addition to this correlation, it is also possible to determine the timing of HIV infection in the individuals
involved in the case, also establishing the temporality of the crime. Two other issues that should also be
considered in these investigations are the quantification of viral load and CD4+ T lymphocytes. With these
measures, it is possible to decrease the chances of an individual contributing to transmission in a criminal
case.
Therefore, through the use of these tools, it is possible to determine whether there is a crime, and what path
the criminal agent takes. Thus, it becomes possible to specify, in this case, authorship and materiality of the
fact, generating greater reliability for the State's duty to punish.
References
1. Fanales-Belasio E, Raimondo M, Suligoi B, Buttò S. HIV virology and pathogenetic mechanisms of
infection: a brief overview. Ann Ist super Sanità. 2013, v. 49, n. 3, p. 255–260.
2. Pa L. Human immunodeficiency viruses and their replication; 3rd. ed. Philadelphia, Lippincott-Raven:
[s.n.], 1996.
3. Tenório LZ, Silva FH, Han SW. A Potencialidade dos Lentivetores na Terapia Gênica. Revista
Brasileira de Clínica Médica. 2008, p. 260–267.
4. Sharp PM, Hahn BH. Origins of HIV and the AIDS pandemic. Cold Spring Harbor Perspectives in
Medicine. 2011, v. 1, n. 1, p. 1–22.
5. Winslow CY, Kedel FA. Human immunodeficiency virus. Dermatological Manifestations of Kidney
Disease. 2015, p. 45–56.
6. Hahn BH, et al. Scientific and Public Health Implications. Science. 2000, v. 287, n. 5453, p. 607–614.
7. Kalish ML, et al. Central African hunters exposed to simian immunodeficiency virus. Emerging
Infectious Diseases. 2005, v. 11, n. 12, p. 1928–1930.
8. Marx PA, Alcabes PG, Drucker E. Serial human passage of simian immunodeficiency virus by unsterile
injections and the emergence of epidemic human immunodeficiency virus in Africa. Philosophical
Transactions of the Royal Society B: Biological Sciences. 2001, v. 356, n. 1410, p. 911–920.
9. Loreto S, Azevedo-Pereira JM. A infecção por HIV–importância das fases iniciais e do diagnóstico
precoce. Acta Farmacêutica Portuguesa. 2012, v. 1, n. 2, p. 5–17.
10. Huynh K, Gulick PG. HIV Prevention. StatPearls, 2019.
11. Brasil - MS. Brasília-DF 2018 Diagnóstico da Infecção pelo HIV em Adultos e Crianças. 2017, p. 85.
12. Tortora G J, Funke BR, Case CL. Microbiologia. 12ª ed. Pearson, USA, 2016.
13. UNAIDS. Desafio Unaids. 2017.
14. Pereira C, Ferrara AP, Tomio AR. Prevenção, tá combinado? Jovens e Adultos Vivendo com HIV/Aids,
Prevenção e Cidadania. 2018.
15. Brasil. Protocolo Clínico e Diretrizes Terapêuticas para Manejo da Infecção pelo HIV em crianças e
adolescentes. 2014. http://www.aids.gov.br/pt-br/tags/publicacoes/protocolo-clinico-e-diretrizes-
terapeuticas.
16. Asts EJ, Hazuda DJ. HIV-1 antiretroviral drug therapy. Cold Spring Harbor Perspectives in
Medicine. 2012 Apr;2(4):a007161. doi: 10.1101/cshperspect.a007161.
17. Jota FA. Os Antirretrovirais Através da História, da Descoberta até os Dias Atuais. 2011.
18. Brasil. Medicamentos antirretrovirais. The New England journal of medicine. 2013, p. 1.
19. Zucchi EM, et al. Da evidência à ação: desafios do Sistema Único de Saúde para ofertar a profilaxia
pré-exposição sexual (PrEP) ao HIV às pessoas em maior vulnerabilidade. Cadernos de Saúde Pública.
2018, v. 34, n. 7, p. 1–16.
REVISTA MEDICINA LEGAL DE COSTA RICA ISSN 2215 -5287 Vol. 36 (1) Marzo 2020
112
20. CDC. Pre-Expousure Prophylaxis (PrEP). http://www.cdc.gov/hiv/risk/prep/index.html
21. Waldman AE. Exceptions: The Criminal Law's Illogical Approach to HIV-Related Aggravated
Assaults. New York Law School, 2011.
22. Furini AAC, et al. HIV/AIDS: relação dos níveis de linfócitos TCD4+ e carga viral com o tempo de
diagnóstigo. Arq. Ciênc. Saúde, 2016, v. 23, n. 4, p. 95-98.
<http://www.cienciasdasaude.famerp.br/index.php/racs/article/view/491>.
doi: https://doi.org/10.17696/2318-3691.23.4.2016.491.
23. Rouxx C, et al. From Forensics to Forensic Science. Current Issues in Criminal Justice. 2012, v. 24, n
1, p.7-24. https://doi.org/10.1080/10345329.2012.12035941
24. Abrantes FEB. Inovações tecnológicas da ciência forense e suas implicações jurídicas. 2014.
www.ambito-
juridico.com.br/site/?n_link=revista_artigos_leitura&artigo_id=19543&revista_caderno=27.
25. Bernard EJ, et al. HIV forensics: Pitfalls and acceptable standards in the use of phylogenetic analysis
as evidence in criminal investigations of HIV transmission. HIV Medicine. 2007, v. 8, n. 6, p. 382–387.
doi: 10.1111/j.1468-1293.2007.00486.x
26. Senés-Guerrero C, Schüssler A. A conserved arbuscular mycorrhizal fungal core-species community
colonizes potato roots in the Andes. Fungal Diversity. 2016, v. 77, n. 1, p. 317–333.
doi: 10.1007/s13225-015-0328-7
27. Kenah E, et al. Molecular Infectious Disease Epidemiology: Survival Analysis and Algorithms Linking
Phylogenies to Transmission Trees. PLoS Computational Biology. 2016, v. 12, n. 4, p. 1–29.
https://doi.org/10.1371/journal.pcbi.1004869
28. Baele G, et al. Emerging concepts of data integration in pathogen phylodynamics. Systematic Biology.
2017, v. 66, n. 1, p. e47–e65. doi: 10.1093/sysbio/syw054.
29. Chang AB, et al. Phylogeny as a guide to structure and function of membrane transport proteins.
Molecular Membrane Biology. 2004, v. 21, n. 3, p. 171–181.
http://www.scielo.sa.cr/scielo.php?script=sci_arttext&pid=S1409-
00152019000200056&lng=en&tlng=es.
30. Carrillo-Araujo M, et al. Phyllostomid bat microbiome composition is associated to host phylogeny and
feeding strategies. Frontiers in Microbiology. 2015, v. 6, n. may, p. 1–9.
31. Matsen FA. Phylogenetics and the human microbiome. Systematic Biology, v. 64, n. 1, p. e26–e41,
2015. https://doi.org/10.1093/sysbio/syu053
32. Caldart ET, et al. Phylogenetic Analysis: Basic Concepts and Its Use as a Tool for Virology and
Molecular Epidemiology. 2016, v. 44, n. Sep, p. 1392, 2016. https://doi.org/10.22456/1679-9216.81158
33. Zweigerdt R. Molecular Phylogenetics: Concepts for a Newcomer. Advances in Biochemical
Engineering/Biotechnology. 2016, p. 1–35. doi: 10.1007/10_2016_49
34. França LTC, et al. A review of DNA sequencing techniques. Quartely Reviews of Biophysics, 2002.
https://doi.org/10.1017/S0033583502003797
35. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Biochemistry.
1977, v. 74. DOI: 10.1073/pnas.74.12.5463
36. Alberts B, et al. Biologia molecular da célula. 6. ed. Porto Alegre: Artmed, 2017.
37. Alvarez-Cubero MJ, et al. Next generation sequencing: an application in forensic sciences? Annals of
Human Biology. 2017, v. 44, n. 7, p. 581–592. doi: 10.1080/03014460.2017.1375155
38. Castro-Nallar E, et al. The evolution of HIV: Inferences using phylogenetics. 2013, v. 2012, n. 2, p.
777–792. doi: 10.1016/j.ympev.2011.11.019
39. Alves BM, et al. Estimating HIV-1 Genetic Diversity in Brazil Through Next-Generation Sequencing.
Frontiers in Microbiology. 2019, v. 10, n. Apr, p. 1–11. https://doi.org/10.3389/fmicb.2019.00749
40. Abecasis AB, Pingarilho M, Vandamme AM. Phylogenetic analysis as a forensic tool in HIV
transmission investigations. Aids. 2018, v. 32, n. 5, p. 543–554. doi: 10.1097/QAD.0000000000001728
REVISTA MEDICINA LEGAL DE COSTA RICA ISSN 2215 -5287 Vol. 36 (1) Marzo 2020
113
41. La, C. et al. Viral diversity from next-generation sequencing of HIV-1 samples provides precise
estimates of infection recency and time since infection Louisa. “Applied Catalysis A, General”. 2019,
n. 2010, p. 1–19. doi: 10.1093/infdis/jiz094
42. Sosin DV, et al. Molecular mechanisms oh HIV-1 Genetic Diversity. Molecular Biology (Mosk), 2017,
51(4):547-560. doi: 10.7868/S0026898417030156
43. Cuevas JM, et al. Extremely High Mutation Rate of HIV-1 In Vivo. PLOS Biology, 2015, 16,
13(9):e1002251. doi: 10.1371/journal.pbio.1002251
44. Pinto ME, Struchiner CJ. A diversidade do HIV-1: uma ferramenta para o estudo da pandemia.
Cadernos de Saúde Pública. 2006, v. 22, n. 3, p. 473–484. http://dx.doi.org/10.1590/S0102-
311X2006000300002
45. Andrews SM, et al. Recent advances in understanding HIV evolution. F1000 Reserch, 2017, 28, 6:597.
doi: 10.12688/f1000research
46. Scaduto DI, et al. Source identification in two criminal cases using phylogenetic analysis of HIV-1
DNA sequences. PNAS. 2010, 14, 107(50), p.21242-7. doi: 10.1073/pnas.1015673107
47. Rambaut A, et al. The causes and consequences of HIV evolution. Nature Reviews Genetics. 2004,
5(1), p.52-61. DOI: 10.1038/nrg1246
48. Faria NR, et al. The early spread and epidemic ignition oh HIV-1 in human populations. Science.
2014, 3, 346(6205). p-56-61. doi: 10.1126/science.1256739
49. Bezemer D, et al. Dispersion of the HIV-1: Epidemic in Men Who Have Sex with Men in the
Netherlands: A Combined Mathematical Model and Phylogenetic Analysis. PLOS medicine. 2015.
https://doi.org/10.1371/journal.pmed.1001898
50. Pineda-Peña AC, et al. Automated subtyping of HIV-1 genetic sequences for clinical and surveillance
purposes: perfomance evaluation of the new REGA version 3 and seven other tools. Elsevier. 2013, 19,
p.337-48. doi: 10.1016/j.meegid.2013.04.032
51. Romero-Severson EO, et al. Phylogenetically resolving epidemiologic linkage. PNAS. 2016, 113 (10),
p.2690-2695. https://doi.org/10.1073/pnas.1522930113
52. Eishleman SH, et al. Analysis of genetic linkage of HIV from couples enrolled in the HIV Prevention
Trials Network 052 trial. The Journal of Infectious Diseases. 2011, 15, 204(12), p.1918-26. doi:
10.1093/infdis/jir651
53. Siljic M, et al. Forensic application of phylogenetic analyses – Exploration of suspected HIV-1
transmission case. Forensic Science International: Genetics. 2017, v. 27, p. 100–105. doi:
10.1016/j.fsigen.2016
54. Puerari DN, PORTES MAA, GARRIDO, RG. Autonomia Da Prova Técnica No Processo. Colloquium
Humanarum, v. 11, n. Especial, p. 459–464, 2015.
55. Giovanelli A, Garrido, RG. A perícia criminal no Brasil como instância legitimadora de práticas
policiais inquisitoriais. Revista LEVS/UNESP. 2011, v. 7, p. 5–24.
http://revistas.marilia.unesp.br/index.php/levs/article/view/1672
56. Garrido RG, Giovanelli A. Ciência Forense Uma Introdução à Criminalística. 2. ed. Rio de Janeiro:
Projeto Cultural, 2015. v. 1. 214p.
57. Learn GH, Mullins JI. The microbial forensic use of HIV sequences. HIV Sequence Compendium 2003,
p. 22–37. https://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/2003/partI/Learn.pdf
58. Landi A, et al. Modelling and control of HIV dynamics. 2007, v. 9, p. 162–168.
DOI:10.1016/j.cmpb.2007.08.003
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